Most upper tract urothelial carcinomas (UTUC) are muscle invasive at the time of diagnosis. Current standard methods for the diagnosis of UTUC are invasive. Urine cytology is the only non-invasive test for detecting UTUC, but its sensitivity is low. A novel non-invasive assay for UTUC detection would improve patient outcome. This study aimed to investigate the mutation of cell-free DNA (cfDNA) in urine supernatant to develop a reliable diagnostic biomarker for UTUC patients. We studied urinary cfDNA from 153 individuals, including 56 patients with localized UTUC, and carried out droplet digital PCR assay for TERT promoter and FGFR3 hotspot mutations. We could detect mutations of TERT C228T in 22/56 (39.3%), TERT C250T in 4/56 (7.1%), and FGFR3 S249C in 9/56 (16.1%) patients. FGFR3 mutation was detected only in ≤pT1 tumors (positive predictive value: 100.0%). In combination with cytology results, the sensitivity was 78.6%, and the specificity was 96.0%. Although these data need to be validated in a larger-scale cohort, mutation analysis of TERT promoter and FGFR3 in urinary cfDNA has the potential to be a non-invasive diagnostic marker and reliable factor for tumor staging.